Protein synthesis is carried out by an elaborate translation complex, which is composed of a ribosome, accessory protein factors as well as mRNA and charged tRNA molecules. Like DNA and RNA synthesis, protein synthesis can be divided into initiation, chain elongation and termination stages. Initiation involves the assembly of the translation complex at the first codon in the mRNA. During polypeptide-chain elongation, the ribosome and associated components move in the 5' to 3' direction along the template mRNA. The polypeptide is synthesized from the N-terminus to the C-terminus. Finally, when synthesis of the protein is complete, the translation complex disassembles in a separate termination step. An important part of this disassembly is the release of the ribosome from the mRNA.
Catalysis of peptide bond formation requires the precise juxtaposition by the ribosome of the acceptor ends of the amino acid-charged tRNA's bound in the peptidyl site (i.e., P site) and aminoacyl site (i.e., A site) of its "active site". This activity represents the essential enzymatic activity of the ribosome and is referred to as the "peptidyl transferase activity," an integral component of the large subunit of all ribosomes characterized to date. Studies of bacterial ribosomes have identified the essential active site constituents of the peptidyl transferase activity as a few ribosomal protein subunits and the 23S rRNA. As the integrity of the latter is essential for enzymatic activity, it is assumed that it plays a direct role in the catalysis of peptide bond formation acting as a so-called ribozyme.
A key step in characterizing the peptidyl transferase reaction was achieved by development of the so-called fragment reaction, wherein the P-site aminoacyl-tRNA is replaced by a small-molecule derivative (e.g., 5'-CAACCA-formyl methionine) and the A-site aminoacyl-tRNA is replaced by puromycin (which mimics the 3' terminus of an aminoacyl-tRNA). During the course of the reaction, which is illustrated diagrammatically in FIG. 1, the amino group of puromycin forms a peptide bond with fMet to yield an fMet-puromycin product. Characterization of this simple reaction, which isolates this phase of the overall translation elongation cycle from other steps (e.g., binding of aminoacyl-tRNA's, release of free tRNA's, and mRNA binding and translocation), allowed for the delineation of the protein and RNA components of the ribosome as well as cofactors required specifically for the peptide bond formation step during elongation. Further studies established the authenticity of the fragment reaction as a valid model of normal peptide bond formation reactions in vivo by demonstrating inhibition of this reaction by antibiotics that have been demonstrated to act at this level in whole cells.
A large number of antibacterial agents, including many in current clinical use, inhibit protein synthesis in bacteria by interfering with essential functions of the ribosome. When ribosomal function is perturbed, protein synthesis may cease entirely or, alternatively, it may be sufficiently slowed so as to stop normal cell growth and metabolism. Differences between the prokaryotic 70S ribosomes (composed of 50S and 30S subunits) and the eukaryotic 80S ribosome (composed of 60S and 40S subunits) underlie the basis for the selective toxicity of many antimicrobial agents of this class. However, a limited subset of this class of antimicrobial agents exhibits some cross-reactivity with the 70S ribosomes of eukaryotic mitochondria. This cross-reactivity probably accounts for the host cells cytotoxicity effects observed with some agents and has limited their use as clinical antimicrobial agents. Other agents (e.g., tetracycline), which affect the function of eukaryotic 80S ribosomes in vitro, are still used clinically to treat bacterial infections as the concentrations employed during antimicrobial therapy are not sufficient to elicit host cell toxicity side-effects.
Moreover, protein biosynthesis inhibitors can be divided into a number of different classes based on differences in their mechanisms of action. The aminoglycoside agents (e.g., streptomycin) bind irreversibly to the 30S subunit of the ribosome, thereby slowing protein synthesis and causing mis-translation (i.e., mis-reading) of the mRNA. The resulting errors in the fidelity of protein synthesis are bacteriocidal, and the selective toxicity of this family of agents is increased by the fact that bacteria actively transport them into the cell. The tetracycline family of agents (e.g., doxycycline) also binds to the 30S ribosome subunit, but does so reversibly. Such agents are bacteriostatic and act by interfering with the elongation phase of protein synthesis by inhibiting the transfer of the amino acid moieties of the aminoacyl-tRNA substrates into the growing polypeptide chain. However, inhibition mediated by the tetracyclines is readily reversible, with protein synthesis resuming once intracellular levels of the agent's decline. Chloramphenicol and the macrolide family of agents (e.g., erythromycin), in contrast, act on the function/activity of the 50S subunit of the ribosome. These agents are bacteriostatic in nature, and their effects are reversible. Finally, puromycin acts as a competitive inhibitor of the binding of aminoacyl-tRNA's to the so-called aminoacyl site (i.e., A-site) of the ribosome and acts as a chain-terminator of the elongation phase as a result of its incorporation into the growing peptide chain.
Shortcomings with previously available assays for peptidyl transferase activity have hampered the search for novel modulators of peptidyl transferase activity. For example, many previously available assays require the use of radioactive compounds and/or suffer from a lack of sensitivity. Moreover, previously available assays for peptidyl transferase activity are not amenable to high throughput screening methods such as are needed to screen large libraries or groups of potential modulators. Thus, there remains a need in the art for new assay methods for identifying modulators of peptidyl transferase activity. The present invention remedies this and other needs.